CD155/TIGIT signalling plays a vital role in the regulation of bone marrow mesenchymal stem cell–induced natural killer–cell exhaustion in multiple myeloma

T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a potential immune checkpoint for natural killer (NK)-cell exhaustion in multiple myeloma (MM), and its ligand, CD155 on bone marrow mesenchymal stem cells (BMSCs), is highly expressed in newly diagnosed MM (NDMM) but expressed at extremely low levels on myeloma cells. CD155/TIGIT signalling is involved in the regulation of BMSC-induced NK-cell exhaustion. formation loop BMSC– myeloma cell interactions. 1–4 NK cells are lymphocytes tumour cells MHC class cells TIGIT lymphocytes an inhibitory immunoglobulin

they did not focus on the inhibitory receptors on NK cells. We re-analysed these sequencing results to explore the expression of TIGIT and CD96 on NK cells, which are both inhibitory receptors. TIGIT was highly expressed in monoclonal gammopathy of undetermined significance (n = 5), low-risk smouldering myeloma (SMM) (n = 3), high-risk SMM (n = 8) and NDMM (n = 7) compared to that in healthy controls (HCs) (n = 9), whereas CD96 expression was not significantly different ( Figure 1).
Next, we used clinical samples to verify the significant high expression of TIGIT in MM (Table S1). TIGIT expression was higher in NDMM (61.46% ± 12.51%) than in the CR (complete response) group (19.52% ± 13.36%) and in HCs (13.80% ± 5.075%) (both p < .01). Expression of the active receptor CD226 was lower in the NDMM group (50.66% ± 13.59%) than in the CR (84.99% ± 8.390%) and HC (86.43% ± 13.26%) groups (both p < .01; Figure 2B,E). Activated receptors (NKG2D and CD107a) and functional biomarkers (IFN-γ and perforin) on NK cells were decreased significantly in NDMM patients ( Figure 2C,E). On NK cells, changes in levels of CD226, TIGIT, NKG2D, CD107a, the function marker perforin and IFN-γ in bone marrow were consistent with those in peripheral blood (Tables S2 and S3). The expression of activated and functional molecules on TIGIT + NK cells was lower than that on TIGIT − NK cells, whereas those on CD226 + NK cells were elevated ( Figure 2D), indicating that TIGIT expression was significantly increased and correlated with NK-cell modulation.
As the interaction between the bone marrow environment and NK cells has been reported, 10 we speculated that the expression of CD155, CD112 and CD113, ligands of TIGIT and CD226, might be expressed on BMSCs and even myeloma cells. Interestingly, the expression of CD155 was greatly upregulated on BMSCs from NDMM patients (95.07% ± 3.644%), but CD112 and CD113 were expressed at very low levels ( Figure 2F)  and S2C). Furthermore, levels of the NK-cell functional molecules perforin and IFN-γ were negatively associated with CD155 expression on BMSCs. The expression of CD155 was closely associated with MM clinical characteristics ( Figure 2H). Considering that the TIGIT ligand CD155 was highly expressed on BMSCs, we speculated that its immunosuppressive functions in NK cells occur via the interaction with CD155 on BMSCs.
Next, we revealed the mechanism through which BMSCs regulate NK cells via CD155/TIGIT using in vitro co-culture ( Figure 3A). After NK cells were amplified with IL-2, expression of the activated markers NKG2D, NKp44, CD69 and NKp30 on NK cells were increased, but these levels were decreased after NK-cell co-culture with BMSCs for 6 days ( Figure S3B). In co-culture systems, TIGIT mAb and TIGIT + CD226 mAb groups showed restored expression of these markers on NK cells and IFNγ secretion was increased in NK cells ( Figure 3B left, C). To verify the changes in NK-cell function, we added the U266 cell line to co-culture systems; apoptosis of U266 gradually increased after adding TIGIT mAbs ( Figure 3B right). We further detected related protein changes in the CD155/TIGIT signalling pathway; here, SHIP-1 expression decreased and Erk phosphorylation increased with TIGIT blockade ( Figure 3D). As TIGIT binds to CD155 with higher affinity and out-competes CD226 for CD155 binding, we purified the agonist CD226 mAb (WO 2020/023312 A1) to activate CD226 while suppressing TIGIT ( Figure S3E). A blocking TIGIT mAb and agonist CD226 mAb were added to BMSC and NK-cell co-culture systems, and we found that the activation of NK cells increased with TIGIT combined with the agonist CD226 mAb and IFN-γ secretion, and U266 cell apoptosis was elevated ( Figure S3C,D,F).

F I G U R E 2 In multiple myeloma (MM) patients, the expression of T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is significantly increased on natural killer (NK) cells, and its ligand CD155, but not CD112 and CD113, is highly expressed on bone marrow mesenchymal stem cells (BMSCs). (A) NK cells were represented as CD3
In summary, we demonstrated that TIGIT was upregulated, but that CD226 was downregulated, on NK cells from NDMM patients. Regarding the TIGIT ligand, CD155, but not CD112 and CD113, was highly expressed on BMSCs but was expressed at low levels on myeloma cells. In vitro coculture results demonstrated that CD115/TIGIT signalling plays a vital role SI in the interaction between BMSCs and NK cells. Blocking TIGIT could greatly restore NK-cell exhaustion, providing a potential avenue for antitumour immunotherapy for MM ( Figure 4).